Automatic analysis device and automatic analysis method

Abstract

An automatic analysis device according to an embodiment includes a light source unit, a reaction container, a detection unit, and a processing unit. The light source unit emits at least two kinds of light having different wavelengths. The reaction container can accommodate a reaction solution obtained by mixing a measurement substance and a reagent. The detection unit receives a first emission light and a second emission light of at least two different wavelengths emitted from the reaction vessel by irradiating the reaction vessel with incident light emitted from the light source unit. The processing unit calculates the concentration of the measurement object on the basis of the signal output from the detection unit. The first emergent light is fluorescent light obtained after the incident light is subjected to wavelength conversion by the reagent. The detection unit has a separation unit for separating the first emitted light and the second emitted light and receiving the light by the light detector. The processing unit calculates the concentration of the measurement object on the basis of at least one of the two output signals output from the detection unit in accordance with the emitted light.

Description

Technical Field

[0001] The embodiments disclosed in this specification and accompanying drawings relate to an automated analytical apparatus and an automated analytical method for analyzing the components of a test sample using an antigen-antibody reaction. Background Technology

[0002] Among the methods for examining specimens using antigen-antibody reactions, fluorescence polarization, which utilizes the polarization properties of fluorescence, is known. Fluorescence polarization involves irradiating a mixture (reaction solution) containing the test sample (analyte) and a fluorescent reagent with linearly polarized excitation light. The intensity of the fluorescence emitted from the reaction solution is measured by polarization decomposition, and the degree of polarization (polarization anisotropy or polarization anisotropy) is evaluated. This anisotropy value is highly sensitive to the rotational motion of the analyte, which depends on the size of the analyte. On the other hand, in antigen-antibody reactions, if the analyte (antigen) is mixed with an antibody-modified reagent, the antigen and antibody specifically react and bind to form agglutinates.

[0003] Therefore, by measuring anisotropy, changes in the size of the analyte (agglutination reaction) can be detected with high sensitivity. The relationship between the size change of the analyte and the measured anisotropy depends on the concentration relationship between the analyte and the reagent. If a calibration curve is obtained beforehand using a known amount of reagent to obtain the relationship between the concentration of the analyte and the measured anisotropy, the concentration of the analyte can be calculated based on the anisotropy measurement results. Patent Document 1 discloses an analytical apparatus using this fluorescence polarization method. On the other hand, Patent Document 2 discloses an apparatus for analyzing agglutination reactions by measuring the intensity of transmitted light and scattered light emitted from the reaction solution. Furthermore, Patent Document 3 discloses an apparatus for performing analysis by simultaneously measuring fluorescence polarization and the intensity of transmitted light or scattered light.

[0004] Existing technical documents Patent documents Patent Document 1: Japanese Patent No. 1692254 Patent Document 2: Japanese Patent No. 6013796 Patent Document 3: Japanese Patent Application Publication No. 2007-120976 Summary of the Invention

[0005] The technical problem that the invention aims to solve According to the fluorescence polarization method disclosed in Patent Document 1, by optimizing the amount of the mixed reagents, it is possible to detect analytes at very low concentrations with high sensitivity. However, under these reagent conditions adjusted for high-sensitivity detection, there is a problem that the measurable concentration range is limited to the low-concentration region. If the concentration of the analyte exceeds its concentration range, the anisotropy value saturates to a certain value regardless of the analyte concentration, resulting in a lack of sensitivity to the analyte concentration. Conversely, if the reagent amount is adjusted to enable the measurement of the high-concentration region, the sensitivity in the low-concentration region decreases. Thus, the fluorescence polarization method suffers from the problem of not being able to simultaneously achieve high sensitivity and a wide measurable concentration range.

[0006] On the other hand, Patent Document 2 discloses an automated analytical method that simultaneously measures transmitted light and scattered light, selecting the optimal measurement method based on the concentration range of the analyte. That is, by combining two measurement methods with different sensitivities, the measurement range can be expanded. However, Patent Document 2 does not disclose fluorescence polarization. Furthermore, as pointed out in Patent Document 2, if it is unknown within which concentration range a measurement method can be measured with high precision, then even combining different measurement methods is of no use.

[0007] Furthermore, Patent Document 3 discloses an immunoassay that simultaneously measures the intensity of fluorescence polarization and transmitted or scattered light to calculate the modification rate of the modified protein. However, in Patent Document 3, a portion of the scattered light that has not undergone wavelength conversion is received by the detector of the fluorescence polarization assay, thus the apparent modified protein is detected at a high level, making it impossible to calculate the modification rate with high accuracy.

[0008] The problem to be solved by the embodiments disclosed in this specification and accompanying drawings is to provide an automated analytical apparatus and method capable of highly sensitively analyzing the concentration of an analyte over a wide range from low to high concentrations. However, the problems to be solved by the embodiments disclosed in this specification and accompanying drawings are not limited to the above-described problems. Technical problems corresponding to the effects of the various configurations shown in the embodiments described below can also be identified as other technical problems.

[0009] Technical solutions for solving technical problems The automated analysis apparatus of this embodiment includes a light source, a reaction vessel, a detection unit, and a processing unit. The light source emits at least two types of light with different wavelengths. The reaction vessel is capable of containing a reaction solution in which the analyte and a reagent are mixed. By irradiating the reaction vessel with incident light emitted from the light source, the detection unit receives at least two different wavelengths of first emitted light and second emitted light emitted from the reaction vessel. The processing unit calculates the concentration of the analyte based on the signal output from the detection unit. The first emitted light is fluorescence after the incident light has been converted by the reagent wavelength. The detection unit has a separation unit for separating the first emitted light and the second emitted light and receiving them by a photodetector. The processing unit calculates the concentration of the analyte based on at least one of two output signals output from the detection unit corresponding to the first emitted light and the second emitted light.

[0010] Invention Effects According to the present invention, an automated analytical apparatus is provided that can analyze the concentration of an analyte with high sensitivity over a wide range of regions from low to high concentrations. Attached Figure Description

[0011] Figure 1 This is a diagram illustrating an example of the measurement range of fluorescence polarization method.

[0012] Figure 2 This is a schematic diagram showing the state of the analyte, fluorophore, and their aggregates in the reaction solution under different concentration conditions.

[0013] Figure 3 This is a schematic diagram showing the changes in the concentration of each signal relative to the analyte obtained through fluorescence polarization and scattering methods.

[0014] Figure 4 This is a schematic diagram of the structure of the photometer section of the automatic analysis device in Embodiment 1.

[0015] Figure 5 This is a detailed diagram of the fluorescence polarization measurement system in Example 1.

[0016] Figure 6 From Figure 5 The spectrum of light emitted by the reaction vessel in the reactor.

[0017] Figure 7 yes Figure 5 The film properties of the dichroic mirror in the image.

[0018] Figure 8 yes Figure 5 The spectrum of light separated by the dichroic mirror.

[0019] Figure 9 yes Figure 5 Other film properties of the dichroic mirror.

[0020] Figure 10 It has Figure 5 The spectrum of light separated by a dichroic mirror for other membrane properties.

[0021] Figure 11 This is a schematic diagram of the reagents, analytes, and their aggregates in Example 1.

[0022] Figure 12 This is a diagram illustrating the measurement and analysis process in Example 1.

[0023] Figure 13 This is a schematic diagram that represents the anisotropic signal based on fluorescence polarization and the polarization-cancelled signal based on scattering as a function of reaction time.

[0024] Figure 14 This is a detailed diagram of another embodiment 1 in Example 1.

[0025] Figure 15 yes Figure 14 The spectrum of light separated by the dichroic mirror.

[0026] Figure 16 It has Figure 14 The spectrum of light separated by a dichroic mirror for other membrane properties.

[0027] Figure 17 This is a detailed diagram of another embodiment 2 in Example 1.

[0028] Figure 18 This is a detailed diagram of another configuration 3 in Embodiment 1.

[0029] Figure 19 This is a detailed diagram of another configuration 4 in Embodiment 1.

[0030] Figure 20 This is a detailed diagram of another embodiment 5 in Example 1.

[0031] Figure 21 This is a detailed diagram of the fluorescence measurement system in Example 2.

[0032] Figure 22 This is a schematic diagram of the automatic analysis device in Example 3.

[0033] Explanation of reference numerals in the attached figures 1. Analyte 2. Fluorescent cells 3, 6 collective 4. Antibodies 5 Scattering particles 10, 110 Light Source Section Light sources 11, 21, 111, 121 Collimating lenses: 12, 22, 41, 112, 122, 141 13, 113 Short-pass filters Polarizing filters 14, 23, 44, 54, 64 15, 55, 155, 115 dichroic mirrors 16, 116 reflectors Condensing lenses: 17, 47, 57, 67, 117, 157, 167 30 Reaction Vessel 31, 131 reaction solution 40, 140 Testing Department 42, 142 Excitation cutoff filters 43 Polarizing Beam Splitter (PBS) 46, 56, 156 bandpass filters 48, 58, 68, 158, 168 detectors 70 Processing Department 200 Analysis Department 210 discs 220a, 220b Metering Section 230 Notes Department 240 First Reagent Dispensing Section 250 Second Reagent Dispensing Section 260 Stirring Section 270 Cleaning and drying section 280 Control Department 290 Memory 300 Display Unit 1000 Automatic Analysis Device. Detailed Implementation

[0034] Hereinafter, the automatic analysis apparatus and automatic analysis method according to the embodiments will be described with reference to the accompanying drawings.

[0035] (The principle of the automatic analysis device and an explanation of the project) The automated analysis apparatus of this embodiment mixes reagents in test samples such as blood and urine collected from human subjects and measures the concentration of the desired test item (analyte) contained in the test sample. The reagents contain a fluorophore (fluorescent molecule), and the fluorophore is modified with an antibody. This antibody specifically reacts with the antigen (analyte) contained in the test sample, causing the reagent containing the fluorophore to agglutinate via the antigen. By quantifying the degree of agglutination, the concentration of the analyte can be determined. Regarding this quantification, in the automated analysis apparatus of this embodiment, the polarization dependence of fluorescence intensity is measured, and the degree of polarization (anisotropy) is calculated to evaluate the degree of agglutination. For example, when a linearly polarized excitation light is irradiated onto the reaction solution, the fluorescence intensity I of the polarized light parallel to the polarization direction of the excitation light is measured for the fluorescence emitted from the reaction solution. para Fluorescence intensity I of orthogonally polarized light orth Using these two measurements, the anisotropy r is calculated according to the following formula (1).

[0036] r=(I para -I orth ) / (I para +2I orth ... (Formula 1) Alternatively, anisotropy r can be calculated according to the following formula (2).

[0037] r=(I para -I orth ) / (I para +I orth ... (Equation 2) Phosphors emit light by absorbing and excitation light according to the relative relationship between the polarization direction of the excitation light and the orientation (molecular axis) of the fluorescent molecules. On the other hand, phosphors undergo rotational and translational motion in the reaction solution through Brownian motion. Here, during the luminescence process, if the rotation of the phosphor is slow enough compared to its fluorescence lifetime to negligiblely affect its effect, light is absorbed and emitted along the molecular axis parallel to the excitation light. Therefore, the fluorescence intensity of polarized light parallel to the excitation light is measured to be maximum. Conversely, if the rotational motion of the phosphor is very high compared to its fluorescence lifetime, the phosphor rotates randomly from light absorption to emission, resulting in unpolarized fluorescence. In an intermediate state, if the fluorescence lifetime and rotational motion are approximately equal, the phosphor emits light while maintaining the polarization direction of the excitation light to some extent, thus exhibiting polarized fluorescence characteristics. Under these conditions, capturing changes in the rotational motion of the phosphor caused by the aggregation reaction by measuring the anisotropy of fluorescence is a major characteristic of fluorescence polarization methods. This rotational motion depends on the volume of the phosphor (e.g., the cube of its size), making it highly sensitive to changes in the phosphor's size. If this principle is utilized, even analytes with very low concentrations can be used to determine the degree of aggregation in the reaction solution with high sensitivity.

[0038] However, since there is an upper limit to the value of this anisotropy r, it is necessary to measure it within the range that does not reach its upper limit. Figure 1 This represents the result of determining the concentration of an analyte using fluorescence polarization. The horizontal axis represents the concentration of the analyte in the reaction solution, and the vertical axis represents the anisotropy r. Figure 1 In the region where anisotropy r varies with the concentration of the analyte (the measurement range in the figure), the measurement result of anisotropy r corresponds one-to-one with the concentration of the analyte, thus allowing the concentration of the analyte to be calculated from the anisotropy r. However, if the anisotropy r exceeds a certain upper limit r... max If the anisotropy value is less than the upper limit r, then the concentration relative to the analyte becomes a fixed value (the saturation region in the figure), and therefore the concentration cannot be calculated based on the anisotropy value. That is, if the anisotropy value r is less than the upper limit r... max Then the concentration can be quantified; if it is r max Above this point, anisotropic saturation occurs, therefore the concentration cannot be determined. Figure 1 In the example, it can be seen that the concentration measurement range is approximately 2 digits. Furthermore, the lower concentration limit of the measurement range is determined by the S / N ratio of the measured fluorescence intensity.

[0039] Here, by adjusting the amount of fluorophore used in the reaction, the detection range can be shifted towards the high concentration side. However, in this case, the detection sensitivity on the low concentration side is sacrificed, and the characteristics of fluorescence polarization cannot be utilized.

[0040] The upper limit value of the anisotropy r mentioned above max The fluorescence intensity is determined by whether the luminescence of the condensed fluorophore or the luminescence of the uncondensed fluorophore (hereinafter referred to as "free fluorophore") contributes more to the measured fluorescence intensity. Figure 2 This diagram schematically illustrates the state of analyte 1 and fluorophore 2 in the reaction solution, as well as their aggregate 3. Here, it is assumed that the rotation (rotational relaxation time) of the free fluorophore 2 is sufficiently short (rotational fast) compared to the fluorescence lifetime of fluorophore 2. That is, in fluorophore 2 alone, the fluorescence is almost unpolarized due to the effect of rotation, and the measured anisotropy is very small.

[0041] Figure 2 (a) represents the state where the amount of fluorophore 2, used as a reagent, is greater than that of analyte 1. In this case, the anisotropy is the sum of the contributions from the emission of free fluorophore 2 and the emission from the emission of aggregated fluorophore (aggregate 3). This state is... Figure 1 The anisotropy measured under the conditions shown varies with the concentration of analyte 1.

[0042] on the other hand, Figure 2 (b) shows that, with a relatively small number of fluorophores 2 compared to analyte 1, the measured fluorescence is emitted approximately in the form of condensate 3. The anisotropy measured under these conditions is approximately the maximum value achievable. This is... Figure 1 The state of the saturation region is shown. In this state, even if analyte 1 becomes even more concentrated, the fluorescence emitted from condensate 3 does not change, therefore the anisotropy value does not change.

[0043] On the other hand, the scattered light from condensation is affected by anisotropy, and the polarization dependence of the scattered light changes. Therefore, by measuring the polarization dependence of the scattered light (e.g., the polarization-reducing component of the scattered light), the condensation reaction can be evaluated. As described in Patent Document 2, the measurement of scattered light (hereinafter referred to as the "scattering method") can be performed with high sensitivity for low concentrations of the analyte compared to the measurement of transmitted light (hereinafter referred to as the "transmission method"). In contrast, in this embodiment, for both the polarization-reducing component of the scattered light and the anisotropy of fluorescence (fluorescence polarization method) based on the scattering method, the concentration ranges that can be measured with high sensitivity for each have been experimentally determined.

[0044] Figure 3 This is a schematic graph based on experimental results, with the horizontal axis representing the concentration of the analyte and the vertical axes representing the signals from the fluorescence polarization method (changes in anisotropy) and the scattering method (changes in the light intensity of the polarization-de-polarized component), respectively. Figure 3 As shown, within the concentration range of region 1, fluorescence polarization method exhibits sensitivity, and the signal converges to... Figure 1 The measurement range shown (anisotropy r) <r max In the region of region 2, the scattering method lacks sufficient sensitivity and therefore cannot obtain an effective signal. Conversely, in the concentration range of region 2 (anisotropy r1 ≤ r...), the scattering method... <r max In this study, fluorescence polarization method, although close to... Figure 1 The saturation region (anisotropy r ≥ r) max However, it is still within the measurement range. Furthermore, the scattering method can gradually confirm the signal changes caused by the agglomeration reaction. Furthermore, in region 3, the fluorescence polarization method reaches a saturation region and cannot measure the concentration change of the analyte. On the other hand, the scattering method can fully confirm the signal change and can accurately measure the concentration of the analyte. Based on this result, low concentrations of the analyte are measured using fluorescence polarization, and after fluorescence polarization reaches a saturation region, the method is switched to scattering. The method with good sensitivity is selected in between, or both results are used together. This allows for the measurement of the analyte concentration over a wide range, up to the region with relatively high concentrations.

[0045] Here, as a scattering method, a determination based on dynamic light scattering (DLS) can also be performed. In the reaction solution, the analyte (scattering particles) and its aggregates move in random directions (translational motion) through Brownian motion. Smaller particles move faster and larger particles move slower. Therefore, if the autocorrelation function is used to evaluate the time variation of the intensity of scattered light emitted from the reaction solution, the former is observed as a short correlation time, and the latter as a long correlation time. Typically, this autocorrelation function can be expressed as an exponential function such as exp(-Γτ) (τ: delay time). If the attenuation coefficient Γ is used, the Γ of the autocorrelation function for short correlation times is large, and conversely, the Γ of the autocorrelation function for long correlation times becomes small. Therefore, by quantifying the agglomeration reaction based on this attenuation coefficient Γ, the concentration of the analyte can also be determined. Regarding this DLS, experiments have confirmed that it can measure concentrations with higher sensitivity than the transmission method and has a wider range of measurable concentrations. As a result, similar to the polarization elimination of scattering, it has been confirmed that it can be combined with fluorescence polarization methods. Therefore, as a scattering method, scattered polarization-eliminated light or DLS can be used. Alternatively, the intensity of scattered light emitted from the reaction solution at a specific angle can be measured.

[0046] Furthermore, by using two antibodies with different affinities to the antigen, labeled with different fluorophores, and measuring the luminescence characteristics of the fluorophores (e.g., changes in luminescence intensity, anisotropy changes) caused by the binding of the antigen-antibody reaction corresponding to the reaction time, the concentration range that can be measured can be expanded. In high-sensitivity measurements at low concentrations, the luminescence signal based on the antibody with high affinity is measured and analyzed, while at high concentrations, the luminescence signal based on the antibody with low affinity is measured and analyzed. In this case, by measuring and analyzing fluorescence signals at least two wavelengths with different luminescence wavelengths, the concentration of the analyte (antigen) from low to high concentrations can be measured.

[0047] As explained above, in this embodiment, by measuring two different wavelengths of light (fluorescence and scattered light, or two different fluorescences) approximately simultaneously, the two signals that vary according to the reaction time are combined and analyzed, thereby determining the concentration of the analyte over a wide range from low to high concentrations.

[0048] [Example 1] Figure 4 This is a schematic diagram of the optical measurement unit of the automatic analysis apparatus in Example 1. A detailed description of the optical system will follow later. The optical measurement unit includes, for example, a light source unit 10, a reaction vessel 30, a detection unit 40, and a processing unit 70. The light source unit 10 is equipped with a light source that emits two wavelengths of light. One wavelength is the wavelength that excites the phosphor contained in the reagent, and the other wavelength is not absorbed by the phosphor. The wavelength of the light source can be appropriately selected according to the excitation wavelength of the phosphor in the reagent. For example, it can be light from the visible band to the near-infrared band with wavelengths from 400 to 1100 nm, or it can be light in the ultraviolet band with wavelengths below 400 nm or light in the infrared band with wavelengths above 1100 nm. As the light source, LEDs and lasers can be used, but monochromatic light with a relatively narrow spectral width is preferred. The light source unit 10 is an example of a "light source unit." That is, the light source unit 10 emits at least two kinds of light with different wavelengths.

[0049] Light emitted simultaneously from two light sources with different wavelengths passes through polarizers (linear polarizers) mounted on the light source unit 10 and is incident as linearly polarized incident light onto the reaction liquid 31 contained in the reaction container 30. Figure 5The reaction solution 31 contains a test sample containing the analyte and a reagent containing a fluorophore modified with an antibody that specifically reacts with the analyte. In the reaction solution 31, agglutination occurs through an antigen-antibody reaction, depending on the size and concentration of the analyte and the fluorophore, the time elapsed after mixing the analyte and reagent (reaction time), and the temperature of the reaction solution. Furthermore, after the test sample and reagent are dispensed, the reaction solution 31 is stirred by a stirring unit (not shown), ensuring uniform dispersion of the analyte and reagent in the reaction solution. The reaction container 30 is an example of a "reaction container." That is, the reaction container 30 is capable of containing the reaction solution containing the analyte and the reagent that specifically reacts with the analyte. Light emitted from the light source 10 enters from the first surface of the reaction container 30 and exits from the second surface opposite the first surface.

[0050] Fluorescence (first emitted light) caused by the excitation of the reagent by incident light and non-excited light (second emitted light) scattered by the reagent are emitted from the reaction vessel 30. These lights are incident on the detection unit 40, where the excitation light is cut off by an excitation light cutoff filter mounted on the detection unit 40. Then, the fluorescence and scattered light are separated by a polarization beam splitter (hereinafter referred to as "PBS") mounted on the detection unit 40 into fluorescence and scattered light of a polarization component parallel to the polarization of the incident light (polarization preservation component), and fluorescence and scattered light of a polarization component orthogonal to the polarization of the incident light (polarization elimination component), respectively, and are received by a detector mounted on the detection unit 40. The received light signals are sent to the processing unit 70, where the anisotropy r is calculated based on the fluorescence intensity, the autocorrelation function is calculated based on the scattered light intensity, and the concentration of the analyte is output by referring to a pre-prepared relationship between the calculation results and the concentration of the analyte (calibration curve). The detection unit 40 is an example of a "detection unit". That is, by irradiating the reaction vessel 30 with incident light emitted from the light source 10, the detection unit 40 receives at least two different wavelengths of first emitted light and second emitted light emitted from the reaction vessel 30. The first emitted light is fluorescence after the incident light has been converted by the reagent wavelength. The detection unit 40 has a separation unit for separating the first emitted light and the second emitted light and receiving the light by a photodetector.

[0051] The processing unit 70 performs its functions, for example, by executing a program stored in memory (not shown) through a hardware processor (computer). A hardware processor may refer to a circuit such as a CPU (Central Processing Unit), GPU (Graphics Processing Unit), Application Specific Integrated Circuit (ASIC), or programmable logic device (e.g., a Simple Programmable Logic Device (SPLD), Complex Programmable Logic Device (CPLD), or Field Programmable Gate Array (FPGA)).

[0052] Processing unit 70 is an example of a "processing unit". Specifically, processing unit 70 calculates the concentration of the analyte based on the signal output from detection unit 40. Processing unit 70 calculates the concentration of the analyte based on at least one of two output signals output from detection unit 40 corresponding to the first emitted light and the second emitted light. Processing unit 70 evaluates the signal change of the output signal after any time elapsed since the start of the reaction in which the analyte and reagent are mixed, and calculates a first signal change based on the first emitted light and a second signal change based on the second emitted light. If the first signal change meets a predetermined condition, the concentration of the analyte is calculated based on the first signal change; otherwise, the concentration of the analyte is calculated based on the second signal change. The reagent includes scattering particles containing fluorescent molecules. The first signal change is a signal change based on fluorescence anisotropy, and the second signal change is a signal change based on an autocorrelation function calculated from the intensity of the scattered light or the time variation of the scattered light intensity. As one of the other signal changes, the reagent includes at least two antibodies with different affinities, each labeled with a fluorescent molecule emitting light at a different wavelength. The first and second signal changes are signal changes based on the fluorescence intensity of the fluorescence at different wavelengths. The specified condition is to compare the changes in the first signal and the changes in the second signal, and calculate the concentration of the analyte based on the signal change that is greater relative to the concentration change of the analyte or the time elapsed.

[0053] (Detailed description of the fluorescence polarization measurement system) use Figure 5 A detailed description of the fluorescence polarization measurement system of Example 1 is provided. The light emitted from the light source 11 is diffused and is substantially parallelized by the collimating lens 12. Here, the light source 11 is an LED that emits excitation light with a wavelength of 340 nm.

[0054] Light passing through the collimating lens 12 passes through the short-pass filter 13, the polarizer 14, and the dichroic mirror 15 (which allows excitation light to pass through and reflects red light), is reflected by the reflecting mirror 16, and is focused by the condenser lens 17 to illuminate the reaction liquid 31 contained in the reaction vessel 30. At this time, the short-pass filter 13 has the characteristic of allowing the excitation wavelength to pass through, and can block unwanted light other than the desired wavelength received by the detection unit 40. Furthermore, the polarizer 14 only allows light to pass through... Figure 5 The z-direction polarized light shown in the diagram is reflected by mirror 16, and the perpendicularly polarized light (y-direction) is incident on the reaction liquid 31. The z-direction is the direction in which the condenser lens 17, the reaction vessel 30, and the collimating lens 41 are arranged sequentially. In other words, the z-direction is the direction in which the light focused by the condenser lens 17 is directed toward the reaction vessel 30. The y-direction is perpendicular to the z-direction. The x-direction is perpendicular to the z-plane.

[0055] The reaction solution 31 contained in the reaction vessel 30 includes the aforementioned test sample, a reagent containing a fluorophore, and their aggregates. When incident light is irradiated, wavelength-converted fluorescence and unconverted excitation light are emitted from the reaction solution 31. Here, the center wavelength of the fluorescence is 611 nm.

[0056] The diffused fluorescence and excitation light are approximately parallelized by the collimating lens 41 and reach the excitation light cutoff filter 42. At this time, the excitation light cutoff filter 42 is configured to allow the desired wavelength received by the detection unit 40 to pass through, and can block the excitation light with a wavelength of 340 nm that has not undergone wavelength conversion. Next, the fluorescence is split into horizontally (x-direction) polarized light and vertically (y-direction) polarized light by the PBS 43. The light incident with s-polarized light relative to the separation surface of the PBS 43 is reflected at the separation surface, passes through the polarizer 44 (which is configured to maximize the transmission of the reflected linearly polarized light), and the bandpass filter 46. It is then focused by the condenser lens 47, and the detector 48 receives the fluorescence intensity I of the polarized component parallel to the incident light's polarization. para Therefore, detector 48 only receives linearly polarized light in the y-direction.

[0057] Additionally, light incident with p-polarized light relative to the separation surface of PBS43 passes through the separation surface, and is transmitted through a polarizer 54 configured to maximize the transmission of linearly polarized light. Fluorescence with a wavelength of 611 nm passes through a tilted dichroic mirror 55, is focused by a condenser lens 57, and a detector 58 receives the fluorescence intensity I of the polarized component orthogonal to the incident light. orth Therefore, detector 58 only receives linearly polarized light in the x-direction.

[0058] The anisotropy r can be calculated using the above equation (1) or (2) based on the light intensity obtained from the two detectors 48 and 58. Here, detectors 48 and 58 use avalanche photodiodes (hereinafter referred to as "APD"), but high-sensitivity light sensors such as photomultiplier tubes (hereinafter referred to as "PMT") and MPPC (Multi-Pixel Photon Counter) can also be used.

[0059] (Detailed description of the scattered light measurement system) Although the light emitted from light source 21 is smaller than that from light source 11, it has some extension and is roughly parallelized by collimating lens 22. Here, light source 21 is a semiconductor laser (hereinafter referred to as "LD") that emits red light with a wavelength of 635 nm.

[0060] The light passing through the collimating lens 22 is transmitted through the polarizer 23, reflected by the dichroic mirror 15, and then reflected by the reflecting mirror 16 in the same manner as the fluorescence polarization measurement system described above. It is then focused by the condenser lens 17 and irradiates the reaction liquid 31 contained in the reaction vessel 30. At this time, the polarizer 23 only allows light polarized in the z-direction to pass through, irradiating the reaction liquid 31 with light polarized in the y-direction as the incident light.

[0061] The incident light is scattered by the reagents and aggregates in the reaction solution 31, and emitted from the reaction vessel 30 as scattered light.

[0062] The diffused scattered light is approximately parallelized by collimating lens 41 and passes through excitation cutoff filter 42. Then, the scattered light, like the fluorescence, is split into horizontally polarized and vertically polarized light by PBS 43. Scattered light incident with s-polarization relative to the separation surface of PBS 43 is reflected at the separation surface; this light becomes useless in the scattered light measurement and is cut off by bandpass filter 46. This is because the detector 48 does not receive light other than the desired fluorescence.

[0063] On the other hand, the scattered light incident with p-polarized light relative to the separation surface of PBS43 passes through the separation surface and then through polarizer 54. Next, the scattered light that has passed through polarizer 54 is reflected by dichroic mirror 55, focused by condenser lens 67, and detector 68 receives the intensity I of the scattered light of the polarization component orthogonal to the incident light. orth Therefore, detector 68 only receives linearly polarized light in the x-direction. Here, detector 68 uses an APD, but other high-sensitivity light sensors could also be used.

[0064] The dichroic mirror 55 is an example of the "separation unit of the detection unit". That is, the dichroic mirror 55 reflects the light on the long wavelength side of two lights with different wavelengths and allows the light on the short wavelength side to pass through. The PBS 43 is an example of the "polarization separation element". That is, the PBS 43 separates the first emitted light that exits from the reaction solution as linearly polarized light into a parallel direction and an orthogonal direction with respect to the polarization direction of the linearly polarized light, and is received by the light detectors respectively.

[0065] (Effective arrangement of the detector for scattered light measurement) Regarding Figure 5 the arrangement of the detector 68, it will be described in detail in combination with the spectrum in the above optical system.

[0066] Figure 6 The spectrum of the light emitted from the reaction vessel 30 is shown. Figure 6 The solid line in is the spectrum of the fluorescence with a central wavelength of 611 nm that has been wavelength-converted by the excitation light from the light source 11, and the dashed line is the scattered light with a central wavelength of 635 nm that is emitted from the light source 21 and scattered by the reaction solution 31. The light of the spectrum of these two combined lights exits from the reaction solution 31.

[0067] Figure 7 Represents the reflection film characteristics of the dichroic mirror 55. Figure 7 The solid line in is the reflectance of the s-polarized light, the dashed line is the reflectance of the p-polarized light, and the dashed line is the average reflectance. The wavelength at which the average reflectance is 50% is called the cut-off wavelength of this dichroic mirror.

[0068] Figure 8 Represents Figure 5 the spectrum of the light separated by the dichroic mirror 55 in. The solid line is the light that passes through the dichroic mirror 55, and the dashed line is the light that is reflected by the dichroic mirror 55. They are respectively condensed by the condenser lenses 57 and 67 and received by the detectors 58 and 68. In the detector 58, the desired light is the fluorescence with a central wavelength of 611 nm, and the scattered light with a central wavelength of 635 nm that may become noise light is not received. Therefore, a high-precision signal can be obtained. In contrast, in the detector 68, the desired light is the scattered light with a central wavelength of 635 nm, and the fluorescence with a central wavelength of 611 nm that may become noise light is not received. Therefore, a high-precision signal can also be obtained here. If weak noise light becomes a problem, although it is not shown in Figure 5 band-pass filters can also be provided respectively before the condenser lenses.

[0069] Thus, the light emitted from the reaction vessel 30 becomes two lights with different wavelengths. However, when the central wavelength on the long wavelength side (635 nm) is set as A, the central wavelength on the short wavelength side (611 nm) is set as B, and the above cut-off wavelength is set as C, it is set to be incident on the dichroic mirror with the characteristic of A - C < C - B as s-polarized light ( Figure 7The optimal optical configuration is one in which a detector is positioned at the reflection location to capture the desired long-wavelength light (635nm). Figure 5 ).

[0070] On the other hand, when the detector for scattered light is placed at the transmission position of the dichroic mirror 55 and the detector for fluorescent polarized light is placed at the reflection position, such as Figure 8 As shown, the detector for scattered light mainly receives only fluorescence, while the detector for fluorescent polarized light mainly receives only scattered light. Therefore, a detector for receiving long-wavelength light needs to be set at the reflection position of the dichroic mirror 55.

[0071] Next, consider in Figure 5 Optical systems using Figure 9 The dichroic mirror is used when the film properties are shown. The condition is AC > CB. In this case, the spectrum of the light separated by the dichroic mirror is as follows: Figure 10 As shown. Because the light received by detector 68 is mixed with fluorescence, a bandpass filter (not shown) is needed to cut off the fluorescence before detector 68.

[0072] (Explanation of the principle of fluorescence polarization measurement) Here, the reagents used in this embodiment will be described. Typically, fluorescent molecules are small in size, making it difficult to measure scattered light with high precision in reagents composed solely of fluorescent molecules. In this regard, as shown below, the reagents in this embodiment have a composition capable of measuring both fluorescence and scattered light. Figure 11 This is a schematic diagram illustrating the reagents used in this embodiment. Figure 11 (a) is the reagent in which fluorescent molecule 2 aggregates inside scattering particle 5. By modifying scattering particle 5 with antibody 4, which specifically reacts with analyte 1, both fluorescent molecules and scattering particles react specifically with analyte 1 to generate agglutinate 6.

[0073] Here, when the aforementioned excitation light is irradiated, the excitation light is absorbed by the fluorescent molecule 2, generating fluorescence. The anisotropy of the fluorescence changes due to the change in the rotational relaxation time of the fluorescent molecules before and after the formation of the condensate 6, thus altering the measured anisotropy. On the other hand, when non-excitation light is irradiated, the non-excitation light does not interact with the fluorescent molecule 2 and is scattered by the scattering particles 5. The polarization characteristics of the incident light change due to the scattering concentration of the reagent, the size and shape of the condensate. That is, the intensity of the polarization-reducing component of the scattered light changes. Thus, in this embodiment, by irradiating the reagent with light of different wavelengths, such as excitation light and non-excitation light, respectively, both measurements can be performed. For example, different wavelengths of light mounted on the light source unit 10 can be simultaneously illuminated, and after mixing the analyte with the reagent, both the anisotropy of the fluorescence and the polarization-reducing component of the scattered light can be measured continuously.

[0074] In addition, as a component of the reagent, it can also be Figure 11 The configuration is shown in (b). Both fluorescent molecule 2 and scattering particle 5 are modified with antibody 4, which specifically reacts with analyte 1. Through antigen-antibody reaction, a condensate 6 is generated, consisting of scattering particle 5 and fluorescent molecule 2 sandwiching analyte 1. The formation of the condensate slows down the rotational relaxation time of the fluorophore, resulting in a change in the anisotropy of the measured fluorescence. Similarly, the polarization characteristics of the scattering also change due to the formation of the condensate. Therefore, with… Figure 11 Similarly, in case (a), this reagent can be used to simultaneously determine the anisotropy of fluorescence and the polarization elimination component of scattered light to evaluate the aggregation reaction.

[0075] Here, the fluorophore 2 of the reagent can be appropriately selected and designed considering its absorption wavelength, emission wavelength, fluorescence efficiency, fluorescence lifetime, and the combination with the antibody 4 that specifically reacts with the analyte 1. In particular, it is preferable to use a fluorophore 2 with a fluorescence lifetime that is approximately the same class as the rotational relaxation time estimated based on the size of the analyte 1 and the size of the aggregate 6. The particles containing the fluorophore are luminescent particles formed by the aggregation of europium complexes, and latex particles with a diameter of 10 to 500 nm can be used, for example. The size and concentration of the scattering particles also affect the anisotropy of fluorescence. Therefore, it is preferable to design the reagent considering the S / N balance between fluorescence polarization and scattering methods.

[0076] then, Figure 12The following describes the measurement and analysis process in this embodiment. First, in step S1, the analyte and reagent are dispensed into reaction vessel 30 to generate reaction solution 31, initiating the agglomeration reaction. Next, in step S2, excitation and non-excitation light are irradiated onto reaction solution 31, and the anisotropy r of fluorescence and the intensity of the polarization-reduced component of scattered light are measured simultaneously. This measurement in S2 is performed continuously at certain time intervals relative to the reaction time from the start of the agglomeration reaction. In step S3, it is determined whether the predetermined reaction time has elapsed. The predetermined reaction time can be set as the time for the agglomeration reaction to fully converge. Alternatively, it can be set to any time (e.g., 5 minutes) within the range where the change in the reaction can be measured. Even assuming that the reaction has not fully converged, the concentration of the analyte can be determined based on the reaction rate by analyzing the signal change within this time. If it is determined in step S3 that the predetermined reaction time has not elapsed, the measurement in S2 is repeated. The number of measurements performed within the reaction time can be arbitrarily set according to the post-measurement analysis.

[0077] If the predetermined reaction time is determined in step S3, the measurement ends, and the process moves to the analysis flow after step S4. First, in step S4, the anisotropy r of the reaction solution 31 is evaluated based on the measurement results of fluorescence polarization. For example, the anisotropy r can be evaluated based on the result of the last measurement within the reaction time. This anisotropy r is compared with a first predetermined value r1. Here, the predetermined value r1 is, for example, equivalent to... Figure 3 The figure shows r1. If the measured anisotropy r is less than r1 ( Figure 3 If the result is in region 1, step S4; otherwise, in step S5, the concentration of the analyte is calculated based on the anisotropy r result and the calibration curve of the fluorescence polarization method. For example, Figure 3 The solid line is the calibration curve (first calibration curve) for fluorescence polarization. Using a standard sample containing a known concentration of the analyte, under the same measurement conditions as in step S1, the relationship between the concentration of the analyte and the anisotropy value is determined, and this is used as the first calibration curve.

[0078] On the other hand, if the anisotropy r is greater than or equal to r1 (step S4; YES), then proceed to step S6, and compare it with the second specified value r. max Compare. The specified value r max For example, equivalent to Figure 3 The r shown max If the measured anisotropy r is less than r max ( Figure 3If the result is in region 2, step S6; or if not, then the concentration of the analyte is calculated in step S7. In step S7, the concentration can also be calculated based on one of the calibration curves using both fluorescence polarization and scattering measurements. Alternatively, based on the measured signal and the results of a pre-measured calibration curve, a more sensitive measurement method can be selected, and the concentration of the analyte can be calculated based on the result of the selected method. For example, here... Figure 3 The dashed line represents the calibration curve (second calibration curve) for the scattering method. The second calibration curve can also be obtained beforehand using standard samples. Figure 3 In region 2, the first calibration curve can be compared with the second calibration curve, and the one with a sufficiently large S / N ratio and a large change in signal relative to the concentration change of the analyte can be selected.

[0079] Here, Figure 13 The graph schematically represents the anisotropy signal based on fluorescence polarization and the light intensity signal of the polarization-removed component based on scattering as a function of reaction time. The condition for proceeding to step S7 is that the anisotropy r determined by fluorescence polarization is r1 ≤ r. <r max The measured anisotropy values ​​are close to... Figure 1 The saturation region shown indicates that, for example, even if the concentrations of the analytes are different (concentration ρ2 > concentration ρ1), it is as follows: Figure 13 (a) and Figure 13 As shown in (b), in fluorescence polarization, almost the same results are obtained relative to the reaction time. On the other hand, fluorescence polarization and... Figure 13 (a) and Figure 13 The results of the scattering method corresponding to (b) are as follows: Figure 13 (c) and Figure 13 As shown in (d). When the concentration of the analyte is low, the signal change cannot be adequately obtained in the scattering method, becoming... Figure 13 As in (c). In this case, according to Figure 13 The concentration can be calculated from the fluorescence polarization method results shown in (a). On the other hand, when the concentration of the analyte is higher than... Figure 13 When (c) is large, for example, Figure 13 As shown in (d), the signal change is fully obtained through the scattering method. In this case, the concentration of the analyte can be calculated based on the measurement results of the scattering method. That is, based on the measurement results of both the fluorescence polarization method and the scattering method, the measurement method with a large signal change, i.e., the sensitivity to the concentration of the analyte, can be selected.

[0080] In addition, the values ​​r1 and r are specified. max It can also be based on the first calibration curve ( Figure 1 or Figure 3 To determine this. Furthermore, r maxThe anisotropy saturation value can be used to determine this. Alternatively, the anisotropy value corresponding to the concentration at which the scattering method's sensitivity exceeds that of the fluorescence polarization method can be set as r. max In this case, it can also be set to r. max =r1, steps S6 and S7 are omitted. In step S4, when r≥r1 (=r max If the result measured in step S6 is r ≥ r, proceed to step S8. Alternatively, if the result measured in step S6 is r ≥ r max (Step S6; YES), then in step S8, the concentration of the analyte is calculated using the scattering method measurement results.

[0081] Basically, fluorescence polarization exhibits higher sensitivity in the low-concentration region compared to scattering. Therefore, to maximize the utilization of this high sensitivity in the low-concentration region of fluorescence polarization, the reagent dispensing volume can be adjusted based on the measurement limit of the lowest concentration of the analyte that can be measured, under the aforementioned apparatus configuration. Furthermore, it is preferable to adjust the dispensing volume so that fluorescence polarization covers the low-concentration region and scattering covers the high-concentration region, with their respective measurement ranges overlapping. Based on this, as a switching point for analysis using both measurement methods, if the anisotropy is within the measurement range ( Figure 3 In region 1 (step S5), fluorescence polarization is preferentially used to calculate the concentration of the analyte. Conversely, if the anisotropy is in a saturated region ( Figure 3 In region 3 (step S8), the scattering method is used. If it is Figure 3 In region 2 (step S7), the method with higher sensitivity between fluorescence polarization and scattering is used within the region 2. In this embodiment, both fluorescence polarization and scattering results can be obtained, and the appropriate method can be selected during the analysis after step S4.

[0082] Furthermore, in the automatic analysis apparatus of this embodiment, a light-receiving system can be added to measure the polarization component (polarization-preserving component) parallel to the polarization of the incident light (=non-excitation light). This is transmitted light that travels straight through without interacting with the reaction liquid 31, and the agglomeration reaction can also be measured as a change in the intensity of the transmitted light. For example, depending on the concentration of the analyte, all three measurement results can be used: fluorescence polarization (fluorescence), scattering (scattered light), and transmission (transmitted light). As pointed out in Patent Document 2, the transmission method has lower sensitivity at low concentrations compared to the scattering method, but it can measure up to high concentrations. Therefore, measurement results based on the three measurement methods can be obtained before step S3, and analysis can be performed according to the switching points based on the pre-determined calibration curve, such as fluorescence polarization for low concentrations, scattering for medium concentrations, and transmission for high concentrations. By combining the three measurement methods, the high-precision measurement range can be further expanded. As an example, the concentration of the analyte can be measured with a dynamic range of 6 bits or more.

[0083] Here, the calibration curve signal can also use the change since the start of the reaction instead of the fluorescence anisotropy *r* and the intensity of the polarization-de-polarized component of the scattered light itself. For example, the fluorescence evaluation can be set as r0 after the reaction has started, and the change in anisotropy from the start of the reaction can be evaluated as Δr (=r-r0) or r / r0. On the other hand, the evaluation of the scattered light can also be set as ΔI (=I-I0) or I / I0. Here, I0 and I are the intensities of the polarization-de-polarized component of the scattered light after the start and thereafter, respectively.

[0084] The following shows a modified example of the detection unit 40 in Embodiment 1.

[0085] (Other methods of Example 1) Figure 14 This illustrates the configuration of the detection unit 40 located downstream of the reaction vessel 30 in another embodiment 1 of Example 1. Up to the polarizer 54... Figure 5 The same applies. Linearly polarized light in the horizontal direction (x-direction) that has passed through polarizer 54 is incident on dichroic mirror 55 as p-polarized light and is separated into colors. The characteristics of dichroic mirror 55 at this time are as follows: Figure 7 As shown. The transmitted fluorescence passes through the bandpass filter 56, is focused by the condenser lens 57, and is received by the detector 58. In contrast, the scattered light reflected by the dichroic mirror 55 is focused by the condenser lens 67 and received by the detector 68.

[0086] Figure 15 express Figure 14The spectrum of the light separated by the dichroic mirror 55. The desired light in detector 58 is fluorescence with a center wavelength of 611 nm, while the scattered light with a center wavelength of 635 nm, which may become noise, is blocked by the bandpass filter 56. On the other hand, the desired light in detector 68 is scattered light with a center wavelength of 635 nm, while the fluorescence with a center wavelength of 611 nm, which may become noise, is sufficiently reduced and not received. Therefore, a high-precision signal can be obtained even without a bandpass filter.

[0087] in addition, Figure 16 This indicates that the characteristics of the dichroic mirror 55 will be changed to... Figure 9 The spectrum of light separated by the dichroic mirror 55 at this time. At this point, the fluorescence and scattered light are separated and incident on each detector; the amount of light that could become noise is small enough that it may not be necessary to set it. Figure 14 The bandpass filter 56 is shown.

[0088] (Other methods of Example 1 2) Figure 17 The configuration of the detection unit 40 located after the reaction vessel 30 in other embodiment 2 of Example 1 is shown. Figure 5 The polarizer 14 is rotated 90° relative to the y-axis and is set so that only the polarized light in the x-direction passes through. The light is reflected by the mirror 16 and irradiates the reaction liquid 31 with the polarized light in the x-direction.

[0089] Light incident on the detection unit 40 is split into horizontally polarized light and vertically polarized light in the PBS 43. Light incident with s-polarization relative to the separation surface of the PBS 43 is reflected at the separation surface and transmitted through a polarizer 44 configured to maximize the transmission of linearly polarized light. Fluorescence with a wavelength of 611 nm is incident as s-polarized light onto the tilted dichroic mirror 55 and transmitted, focused by the condenser lens 47, and received by the detector 48. At this time, the characteristics of the dichroic mirror 55 are as follows... Figure 7 As shown. In contrast, the scattered light reflected by the dichroic mirror 55 is focused by the condenser lens 67 and received by the detector 68.

[0090] The spectrum separated by dichroic mirror 55 is approximately the same as Figure 8 As shown, the noise in each detector is weak. The characteristics of the dichroic mirror 55 become... Figure 9 In this case, the spectrum is roughly the same as Figure 10 As shown, a bandpass filter or longpass filter with a cutoff wavelength of 611nm for fluorescence needs to be set at the reflection position of the dichroic mirror 55.

[0091] (Other methods of Example 1, 3) Figure 18 This illustrates the configuration of the detection unit 40 located after the reaction vessel 30 in another embodiment 3 of Example 1. Regarding the light path of the reflected PBS 43, up to the polarizer 44... Figure 17 Same.

[0092] Fluorescence with a wavelength of 611 nm is incident on the dichroic mirror 55 arranged obliquely as p-polarized light and passes through it. It passes through the band-pass filter 46, is condensed by the condenser lens 47, and is received by the detector 48. At this time, the characteristics of the dichroic mirror 55 are as Figure 7 shown. In contrast, the scattered light reflected by the dichroic mirror 55 is condensed by the condenser lens 67 and received by the detector 68.

[0093] The spectrum separated by the dichroic mirror 55 is approximately the same as the Figure 15 spectrum shown. Therefore, it is necessary to cut off the scattered light of 635 nm through the band-pass filter 46. When the dichroic mirror 55 has the characteristic of A - C > C - B among the above wavelengths as Figure 9 shown, the light of p-polarized light incident on the dichroic mirror 55 is separated, and the separated spectrum is approximately the same as the Figure 8 spectrum shown, and the noise in each detector is weak.

[0094] (Another Mode 4 of Example 1) Figure 19 The structure of the detection unit 40 located after the reaction vessel 30 in another mode 4 of Example 1 is shown. Up to the excitation light cut-off filter 42, it is the same as Figure 5 the above.

[0095] Scattered light with a wavelength of 635 nm is reflected by the dichroic mirror 55 arranged obliquely, passes through the polarizer 64, is condensed by the condenser lens 67, and is received by the detector 68. Here, since the desired polarized light in the detector 68 is light polarized in the x direction, it is set to a configuration in which light polarized in the z direction does not pass through the polarizer 64. At this time, when using the dichroic mirror with the characteristics shown in Figure 7 the spectrum separated by the dichroic mirror 55 is approximately the same as the Figure 8 spectrum shown. Next, the fluorescence passing through the dichroic mirror 55 is divided into horizontally polarized light and vertically polarized light by the PBS 43, and is respectively received by the detectors for performing fluorescence polarization measurement.

[0096] As described above, it is necessary to provide a detector for receiving light on the long wavelength side at the reflection position of the dichroic mirror 55. In addition, the light emitted from the reaction vessel 30 becomes two lights with different wavelengths. However, when the central wavelength on the long wavelength side is set as A, the central wavelength on the short wavelength side is set as B, and the above cut-off wavelength is set as C, it is preferable to use a dichroic mirror with the characteristic of A - C < C - B when incident on the dichroic mirror with s-polarized light, and use a dichroic mirror with the characteristic of A - C > C - B when incident with p-polarized light.

[0097] (Another Mode 5 of Example 1) Figure 20 This illustrates the configuration of the detection unit 40 located after the reaction vessel 30 in another embodiment 5 of Example 1. Regarding the light path transmitted through the PBS 43, up to the polarizer 54... Figure 5 same.

[0098] Linearly polarized light in the horizontal direction (x direction) that passes through polarizer 54 is focused by condenser lens 57 and received by detector 58. Conversely, light reflected by PBS43 is linearly polarized light in the vertical direction (y direction) that passes through polarizer 44, is focused by condenser lens 47 and received by detector 48.

[0099] Here, fluorescence and scattered light are separated in PBS43 only by their different polarization and received by the detector. Therefore, the light intensity I obtained by simultaneously illuminating light sources of different wavelengths is... para I orth It is the intensity of fluorescence I Fpara I Forth and the intensity of scattered light I Spara I Sorth The results obtained by adding them separately cannot be separated for measurement. Therefore, the light source 11 and the light source 21 are controlled to be lit alternately or sequentially, and the fluorescence and scattered light are measured by separating them with individual detectors accordingly.

[0100] For example, when the light source 11 is first lit, fluorescence is emitted from the reaction vessel 30, and the intensity I obtained by the detectors 48 and 58 can be used to determine the fluorescence intensity. Fpara I Forth The anisotropy is determined. Next, when light source 21 is lit, scattered light is emitted from reaction vessel 30, and the scattered component becomes the light intensity I obtained by detector 58. Sorth Since detector 58 receives fluorescence and scattered light alternately or sequentially, it is possible to acquire both fluorescence polarization and scattering signals approximately simultaneously. The PBS used here corresponds to different wavelengths of fluorescence and scattered light, enabling efficient polarization separation of s-polarized and p-polarized light.

[0101] At this point, depending on the conditions of the reagents contained in the reaction solution 31 and the concentration of the analyte, the intensity difference between the fluorescence or scattered light received by the detector increases, and the light intensity of one side of the detector may sometimes saturate. Therefore, the output of the light source that causes saturation should be smaller than that of other light sources. Alternatively, saturation can be prevented by shortening the illumination time of the light source that causes saturation. Generally, as the concentration increases, the output of scattered light tends to increase, so the output of light source 21 is usually reduced.

[0102] As described above, the light sources 11 and 21 are lit in sequence, and the fluorescence and scattered light are alternately or sequentially received by the detector. At this time, the signals generated based on the light intensities of the fluorescence and the scattered light are alternately or sequentially scanned and output from the detector.

[0103] As another method, an array sensor such as a CCD or CMOS can also be used as the detector. Each light-receiving area of the array sensor is equipped with an RGB color filter, a wavelength cut-off filter, or a light reduction filter, and can simultaneously acquire lights with different wavelengths emitted from the reaction solution. In this case, a light reduction filter can also be arranged for each wavelength, so the above control is not required.

[0104] In this embodiment, LEDs and LDs are used as the light sources, but an SLD (Super Luminescent Diode) with a higher output than the LED, a solid laser that emits YAG harmonics suitable for the spectral width of DLS, a gas laser such as a He-Ne laser, etc., which are suitable for the excitation wavelength of the emission wavelength of the phosphor, can also be used.

[0105] It can also be that regarding the desired light received by the optical detector, when the wavelength at which the reflectivity of the dichroic mirror becomes 50% is set as the cut-off wavelength C, the wavelength of the long-wavelength light in the two different wavelengths of light is set as A, and the wavelength of the short-wavelength light is set as B, if A - C < C - B, s-polarized light is incident on the dichroic mirror for color separation, and if A - C > C - B, p-polarized light is incident on the dichroic mirror for color separation. It can also be that among the light intensities of the optical detector that receives the first emitted light and the optical detector that receives the second emitted light, the output of the first light source that forms the emitted light with a smaller light intensity is larger than the output of the second light source, or the gain or exposure time of the optical detector that receives the emitted light with a smaller light intensity is larger than that of the other optical detector.

[0106] [Embodiment 2] (Fluorescence x Fluorescence) Figure 21 This shows the configuration of the automatic analysis device in Embodiment 2. The configuration of the optical system is substantially the same as that in Embodiment 1, but two lights with different wavelengths emitted from the light source unit 110 are irradiated onto the reaction solution 131.

[0107] The reaction solution 131 consists of two antibodies with different antigen affinities and different fluorescence labels corresponding respectively, and each phosphor emits fluorescence with different wavelengths according to the above two excitation lights. In this embodiment, the changes in the emission intensities of the two phosphors are measured by utilizing the differences in the bindings of the antigen-antibody reactions corresponding to the reaction time. For example, the following method can also be applied: the distance between the reagents is made close through an agglutination reaction to cause energy transfer, and the concentration of the analyte is measured by measuring the change in the intensity of the fluorescence (light enhancement or extinction) generated thereby. Here, according to the measurement method, the reagents are mixed under appropriate conditions.

[0108] The following is a detailed description of a measurement system for determining the concentration of an analyte based on the changes in the luminescence intensity of two different phosphors.

[0109] (Detailed description of the first fluorescence (short wavelength side) measurement) The light emitted from the light source 111 is collimated approximately by the collimating lens 112. Here, as an example, the light source 111 is an LED with a wider spectral width than a laser. The light passing through the collimating lens 112 and the short-pass filter 113 passes through the dichroic mirror 115, is reflected by the reflecting mirror 116, and is focused by the condenser lens 117 to irradiate the reaction liquid 131 contained in the reaction vessel 30. At this time, the short-pass filter 113 has the characteristic of allowing the excitation wavelength to pass through, and can block unwanted light other than the desired wavelength light received by the detection unit 140 emitted by the LED.

[0110] A first fluorescence corresponding to the excitation wavelength is emitted from the reaction vessel 30. This becomes the shorter wavelength side of the two fluorescencees with different wavelengths. The diffusely emitted fluorescence is collimated approximately by the collimating lens 141 and passes through the excitation cutoff filter 142. Then, the fluorescence passes through the tilted dichroic mirror 155 and the bandpass filter 156, is focused by the condenser lens 157, and is received by the detector 158.

[0111] (Detailed description of the second fluorescence (long wavelength side) measurement system) The light emitted from the light source 121 is collimated approximately by the collimating lens 122. Here, as an example, the light source 121 is an LD with a relatively narrow spectral width. The light passing through the collimating lens 122 is reflected by the dichroic mirror 115, and then, as described above, by the reflecting mirror 116, and then focused by the condenser lens 117 to irradiate the reaction liquid 131 contained in the reaction vessel 30.

[0112] A second fluorescence corresponding to the excitation wavelength is emitted from the reaction vessel 30. This becomes the longer-wavelength side of the two fluorescencees with different wavelengths. The emitted second fluorescence, which is extended, is the same as the first fluorescence, is collimated approximately by the collimating lens 141, and passes through the excitation cutoff filter 142. It is then reflected by the dichroic mirror 155, focused by the condenser lens 167, and received by the detector 168.

[0113] (Preparation effective for fluorescence measurement) right Figure 21The configuration after the dichroic mirror 155 will be described in detail. The light emitted from the reaction vessel 130 becomes two fluorescences of different wavelengths. When the center wavelength of the second fluorescence is set as A, the center wavelength of the first fluorescence is set as B, and the wavelength when the average reflectance of s-polarized light and p-polarized light in the reflection film characteristics of the dichroic mirror is 50% is set as the cut-off wavelength C, it is considered to be set in such a way that light is incident on a dichroic mirror having the characteristic of A - C < C - B, and an optical configuration in which a detector for obtaining the second fluorescence is provided at the reflection position ( Figure 21 ).

[0114] The light of the wavelength desired by the detector 158 is the first fluorescence, and the second fluorescence that may become noise light is mixed in, so it is necessary to remove the unnecessary second fluorescence in the band-pass filter 156. In contrast, the light of the wavelength desired by the detector 168 is the second fluorescence, and the first fluorescence that may become noise light is weak, so it is not necessary to provide a band-pass filter here, and a high-precision signal can be obtained. Nevertheless, if weak noise light becomes a problem, although not shown in Figure 21 a band-pass filter for cutting off the weak first fluorescence can also be provided before the condenser lens 167.

[0115] On the other hand, in the case where a detector for obtaining the second fluorescence is provided at the transmission position of the dichroic mirror 155 and a detector for obtaining the first fluorescence is provided at the reflection position, in the detector provided at the reflection position, mainly only the second fluorescence is received, and the desired first fluorescence is not received. Therefore, it is necessary to provide a detector for receiving the second fluorescence at the reflection position of the dichroic mirror 155.

[0116] Next, consider the case when a dichroic mirror having a film characteristic satisfying the condition of A - C > C - B is used in the Figure 21 optical system. In this case, since the first fluorescence is mixed in the light received by the detector 168, it is necessary to provide a band-pass filter (not shown) for cutting off the first fluorescence before the detector 168.

[0117] As described above, when a detector for receiving long-wavelength light is provided at the reflection position of the dichroic mirror 155 and short-wavelength light as noise light is mixed in the received light, a band-pass filter for cutting off the short-wavelength light is provided before the detector.

[0118] (Another aspect of Example 2) As described above, a method for measuring the concentration of an analyte based on the change in the emission intensity of two different phosphors has been described, but it may also be a method for measuring the concentration of an analyte from the polarization anisotropy of two different phosphors. Alternatively, it may be a method for measuring the concentration of an analyte in which one is based on polarization anisotropy and the other is based on the change in the emission intensity of a phosphor.

[0119] The former method, which determines the concentration of the analyte based on the polarization anisotropy of two different fluorophores, is the same as... Figure 5 The optical system has a similar configuration, but in order to measure the polarization anisotropy of two different phosphors, a dichroic mirror needs to be placed at the PBS reflection position, just like the PBS transmission position, to separate the fluorescence of different wavelengths. At this time, a detector is needed to receive the reflected light of the separated light. However, as described in other aspects 5 of Example 1, by applying control to alternately or sequentially illuminate light sources of different wavelengths, the number of detectors can be reduced, and the optical system can be simplified.

[0120] The latter method, which determines the concentration of the analyte based on polarization anisotropy on one side and changes in the luminescence intensity of the phosphor on the other, is similar to... Figure 19 The optical systems have roughly the same configuration, with one side exhibiting polarization anisotropy based on the intensity I received by detectors 48 and 58, respectively. para I orth The change in the luminescence intensity of the other phosphor is calculated and measured by detector 68. In this case, polarizer 64 is not required before detector 68, and polarizer (not shown) is not required after the light source that excites the fluorescence received by detector 68.

[0121] In this embodiment, LEDs and LDs are used as light sources, but SLDs with higher output than LEDs, solid-state lasers emitting YAG harmonics, gas lasers such as He-Ne, which have an excitation wavelength suitable for phosphors, can also be used.

[0122] [Example 3] (Automatic analysis device with rotating disc) Figure 22 This is a configuration diagram of the automatic analysis apparatus 1000 in this embodiment. As disclosed in Japanese Patent No. 5908954, the automatic analysis apparatus 1000 includes an analysis unit 200 and a control unit 280 that controls the analysis unit 200. The control unit 280 controls the measurement process in the photometer unit, receives signals output from the photometer unit, controls the processing unit 70 and the memory 290, and performs data transmission, processing, and storage. In addition, the automatic analysis apparatus 1000 includes a display unit 300 that displays the results processed by the processing unit 70.

[0123] The control unit 280 performs its functions, for example, by executing a program stored in the memory 290 using a hardware processor (computer). The hardware processor includes, for example, a CPU. The control unit 280 is an example of a "control unit." Specifically, the control unit 280 simultaneously, alternately, or sequentially illuminates at least two light sources that emit light of different wavelengths. The detection unit receives the first emitted light and the second emitted light simultaneously, alternately, or sequentially, depending on the illumination time of the light sources.

[0124] The memory 290 is implemented, for example, by semiconductor memory elements such as RAM (Random Access Memory), flash memory, hard disks, optical disks, etc. The memory 290 stores, for example, various data related to the measurement (calibration curves, etc.). This data may also be stored outside the memory 290 (or, based on the memory 290), in an external memory that the automatic analysis device 1000 can communicate with. The external memory receives read and write requests, for example, through a cloud server that manages the external memory, and is controlled by the cloud server.

[0125] The display unit 300 may be, for example, a liquid crystal display, a CRT (Cathode Ray Tube), or an organic EL (Electroluminescence) display. The display unit 300 may also be a display device (e.g., a tablet terminal) capable of wirelessly communicating with the automatic analysis device 1000.

[0126] The analysis unit 200 includes, for example, a rotatable disk 210 and multiple reaction containers 30 arranged on the circumference of the disk 210. The photometric unit may also consist of a photometric unit 220a performing fluorescence polarization and a photometric unit 220b performing scattering. Furthermore, the analysis unit 200 includes a first dispensing unit 230 for dispensing samples such as standard samples and analytes into the reaction containers 30, a first reagent dispensing unit 240 for dispensing a first reagent that reacts with the components contained in the sample, and a second reagent dispensing unit 250 for dispensing a second reagent paired with the first reagent. Additionally, the analysis unit 200 includes a stirring unit 260 for stirring the mixture of sample and reagent, and a cleaning and drying unit 270 for drawing the mixture after measurement from the reaction containers 30 and cleaning and drying the inside of the reaction containers 30. Therefore, the automatic analysis apparatus 1000 can continuously perform a series of processes—dispensing, stirring, measuring, drawing, cleaning, and drying—while rotating the disk 210. In addition, the reaction vessel 30 is housed in a constant temperature bath, and the temperature of the reaction liquid is kept constant.

[0127] In the above-described device configuration, according to Figure 12 The process shown is used to perform measurement and analysis. In this embodiment, measurements are performed in the reaction vessel 30 by timing each photometer 220a and 220b. The measurement is performed multiple times as the disk 210 rotates, as a function of reaction time, to evaluate anisotropy and the polarization elimination component of scattering. As in this embodiment, fluorescence polarization and scattering methods can also be measured approximately simultaneously and independently. In addition, the switching point between fluorescence polarization and scattering methods in the post-measurement processing, and the calibration curves corresponding to each measurement method are stored in the memory 290, and the control unit 280 follows the... Figure 12 The process involves reading out the conditions as needed and executing them.

[0128] Here, the photometer may also include a photometer 22c (not shown) that performs the transmission method. In addition, as described in Embodiment 1, it is considered to combine fluorescence polarization method and scattering method as one photometer 220a, and perform transmission method and other combinations by photometer 220b.

[0129] Thus, this invention preferably utilizes reagents that can be measured by both fluorescence polarization and scattering methods, and adjusts the reagent dosage in a manner where their respective measurement ranges overlap. As an effect of this invention, by appropriately selecting and resolving multiple measurement results, including those from scattering and transmission methods, while maintaining high sensitivity based on fluorescence polarization, it is possible to… Figure 12 The process shown measures a wide range of concentrations in a single procedure. Here, scattering is used to measure the scattered light, and as explained so far, several methods are considered, including the polarization-de-polarization component of the unexcited light, the intensity of light emitted at a certain scattering angle, or the autocorrelation function calculated from the time variation of the scattered light intensity. Alternatively, the intensity of transmitted light based on transmission methods may also be included.

[0130] The objective of this invention is to expand the range of analyte concentrations by combining a method that measures agglutination caused by antigen-antibody reactions as a change in rotational motion using fluorescence polarization and a method that measures changes in scattering cross-sectional area or translational motion using scattering. The former is volume-dependent, and therefore, in principle, is more sensitive to minute changes compared to the latter. Utilizing this principle, agglutination changes caused by antigen-antibody reactions can be measured independently using methods based on two different physical phenomena. Furthermore, by appropriately selecting the two measurement results according to the concentration of the analyte, the analyte range can be expanded.

[0131] The embodiments of the present invention have been described above with reference to exemplary embodiments, but it should be understood that the present invention is not limited to the embodiments described above. The scope of the claims should be interpreted in the broadest sense, encompassing all such modifications and equivalent constructions and functions.

Claims

1. An automatic analysis device, characterized in that, have: The light source emits at least two types of light with different wavelengths; A reaction vessel capable of containing a reaction solution consisting of a mixture of an analyte and a reagent that specifically reacts with the analyte; The detection unit receives first emitted light and second emitted light of at least two different wavelengths emitted from the reaction vessel by irradiating the reaction vessel with incident light emitted from the light source unit; as well as The processing unit calculates the concentration of the analyte based on the signal output from the detection unit. The first emitted light is fluorescence, which is the incident light whose wavelength has been converted by the reagent. The detection unit includes a separation unit for separating the first emitted light and the second emitted light and receiving them with a photodetector. The processing unit calculates the concentration of the analyte based on at least one of two output signals output from the detection unit corresponding to the first emitted light and the second emitted light.

2. The automatic analysis device according to claim 1, characterized in that, The processing unit, The signal change of the output signal after any time elapsed since the start of the reaction in which the analyte and the reagent were mixed is evaluated. Calculate the first signal change based on the first emitted light and the second signal change based on the second emitted light, respectively. If the change in the first signal meets a certain specified condition, the concentration of the analyte is calculated based on the change in the first signal; if the specified condition is not met, the concentration of the analyte is calculated based on the change in the second signal.

3. The automatic analysis device according to claim 2, characterized in that, The reagent contains scattering particles that encapsulate fluorescent molecules. The first signal change is a signal change based on fluorescence anisotropy. The second signal change is a signal change based on the autocorrelation function calculated from the intensity of the scattered light or the time variation of the scattered light intensity.

4. The automatic analysis device according to claim 2, characterized in that, The reagent comprises at least two antibodies with different affinities, and each antibody is labeled with a fluorescent molecule that emits light at a different wavelength. The first signal change and the second signal change are signal changes based on the fluorescence intensity of fluorescence at different wavelengths.

5. The automatic analysis apparatus according to any one of claims 1-4, characterized in that, The separation unit of the detection section is a dichroic mirror that reflects light from the longer wavelength side and transmits light from the shorter wavelength side of two different wavelengths.

6. The automatic analysis apparatus according to any one of claims 1-3, characterized in that, The light source has a linear polarizer, and at least one type of light emitted from the light source irradiates the reaction liquid as linearly polarized light. The detection unit has a polarization separation element that separates the polarization direction of the first emitted light emitted from the reaction liquid through the linear polarization into a parallel direction and an orthogonal direction, which are respectively received by the photodetector.

7. The automatic analysis device according to claim 5, characterized in that, When the wavelength with a reflectivity of 50% of the dichroic mirror is set as the cut-off wavelength C, the wavelength of the longer-wavelength light among the two different-wavelength lights is set as A, and the wavelength of the shorter-wavelength light is set as B, if A - C < C - B, the desired light received by the light detector is incident on the dichroic mirror as s-polarized light for color separation, and if A - C > C - B, the desired light received by the light detector is incident on the dichroic mirror as p-polarized light for color separation.

8. The automatic analysis device according to any one of claims 1-4, characterized in that Among the light intensity of the light detector receiving the first emitted light and the light intensity of the light detector receiving the second emitted light, The output of the first light source that forms the emitted light with a small light intensity is greater than the output of the second light source, or the gain or exposure time of the light detector receiving the emitted light with a small light intensity is greater than that of the other light detector.

9. The automatic analysis device according to any one of claims 1-4, characterized in that The automatic analysis device further has a control unit that alternately or sequentially lights at least two light sources that emit light with different wavelengths, The detection unit alternately or sequentially receives the first emitted light and the second emitted light according to the lighting time of the light source.

10. The automatic analysis device according to any one of claims 1-4, characterized in that The light emitted from the light source unit is incident from the first surface of the reaction vessel and exits from the second surface opposite to the first surface.

11. The automatic analysis device according to claim 2 or 3, characterized in that The specified condition compares the first signal change with the second signal change, and calculates the concentration of the analyte based on the signal change of the larger one with respect to the concentration change of the analyte or the passage of time.

12. An automatic analysis method, characterized in that, Comprising: An irradiation step of irradiating a reaction solution mixed with an analyte and a reagent that specifically reacts with the analyte with at least two lights with different wavelengths; A light-receiving step of separately separating and receiving at least two first emitted lights and second emitted lights with different wavelengths emitted from the reaction solution; and A processing step of calculating the concentration of the analyte based on the signals output by the light-receiving step, In the processing step, the concentration of the analyte is calculated based on at least any one of the change in the signal based on the first emitted light and the change in the signal based on the second emitted light.

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